Beneficial base substitutions in Escherichia coli fucO gene for enhancement of glycolic acid production.

Microbial production of glycolic acid (GA) from ethylene glycol is extensively used in a variety of industries because ethylene glycol is not only an inexpensive raw material but also the main component of industrial wastes. In this study, we produced GA from ethylene glycol using Escherichia coli overexpressing the endogenous 1,2-propanediol oxidoreductase (fucO) and lactaldehyde dehydrogenase (aldA) genes. To increase GA productivity, we screened a random mutant library generated using an error-prone polymerase chain reaction of fucO and obtained FucO mutants MF2-9 and MF6-9 with enhanced GA production in Lysogeny Broth medium containing 800 mM ethylene glycol. MF2-9 contained three amino acid substitutions (D23E, E222K, and G363S) and two synonymous mutations (coding DNA [c.] 93G > A and c.1131T > C) in fucO. MF6-9 contained one amino acid substitution (L377H) in FucO. An amino acid substitution (L377H) and a single synonymous mutation (c.1131T > C) in fucO contributed to the enhancement in GA production. Notably, cell lysates from E. coli harboring a synonymous mutation (c.1131T > C) or amino acid substitution (L377H) in fucO showed that only AldA activity was 1.3-fold higher than that of the cell lysate from E. coli harboring the wild-type fucO. We confirmed that c.1131T > C and L377H mutations increased aldA expression in E. coli. Analysis of mRNA levels and simulation of mRNA stabilization indicated that base substitutions at positions c.1130T, which corresponds to L377H amino acid substitution, and c.1131T increased aldA expression due to partial destabilization of the mRNA. These findings will be useful for the large-scale microbial production of GA from industrial waste.

How effective are mood stabilizers in treating bipolar patients comorbid with cPTSD? Results from an observational study.

BACKGROUND: Multiple traumatic experiences, particularly in childhood, may predict and be a risk factor for the development of complex post-traumatic stress disorder (cPTSD). Unfortunately, individuals with bipolar disorder (BP) are more likely to have suffered traumatic events than the general population. Consequently, cPTSD could be comorbid with BD, and this may negatively affect psychopathological manifestations. To date, no one has explored whether such comorbidity also affects the response to treatment with mood stabilizers in BD patients. RESULTS: Here, a cross-sectional study was carried out by comparing the response to treatment, measured by the Alda scale, in a cohort of 344 patients diagnosed with BD type I and II, screened for the presence (or absence) of cPTSD using the International Trauma Questionnaire. The main result that emerged from the present study is the poorer response to mood stabilizers in BD patients with comorbid cPTSD compared with BD patients without cPTSD. CONCLUSIONS: The results collected suggest the need for an add-on therapy focused on trauma in BD patients. This could represent an area of future interest in clinical research, capable of leading to more precise and quicker diagnoses as well as suggesting better tailored and more effective treatments.

Alda-1 ameliorates air embolism-induced acute lung injury.

OBJECTIVE: Evidence suggests that aldehyde dehydrogenase 2 (ALDH2) offers protection against damage caused by oxidative stress in diverse rodent models. Nonetheless, the effect of Alda-1, a compound that activates ALDH2, on acute lung injury (ALI) induced by air embolism (AE) remains unclear. The objective of this study was to explore the protective effects of Alda-1 in ALI induced by AE. METHODS: A rat model of in situ isolated perfused lung was established to investigate AE-induced ALI. Air was infused into the pulmonary artery at 0.25 mL/min for 1 minute. Before inducing AE, different doses (10, 20, or 30 mg/kg) of Alda-1 were given through intraperitoneal injection. Pathological changes in lung tissue were assessed using hematoxylin-eosin staining. We performed Western blot analysis to assess the protein levels of ALDH2,4-hydroxy-trans-2-nonenal (4-HNE), Bcl-2, caspase-3, phosphatidylinositol 3-kinase (PI3K), Akt, IκB-α, and nuclear NF-κB. RESULTS: Notably, AE results were demonstrated as harmful to the lungs, which is evidenced by intensified lung edema and disruption of lung tissue structure. Furthermore, AE caused a decrease in ALDH2 expression, increased accumulation of 4-HNE and MDA, infiltration of neutrophils, increased production of inflammatory cytokines, apoptosis, and upregulation of the PI3K/Akt and NF-κB signaling pathways within the lungs. Administration of a 20 mg/kg dose of Alda-1 alleviated the detrimental effects induced by AE. CONCLUSION: Alda-1 shows promise in mitigating AE-induced ALI, possibly through the upregulation of ALDH2 expression and suppression of the PI3K/Akt and NF-κB signaling pathways. Further research is warranted to validate these findings and to explore their translational potential in human subjects.

Impact of common ALDH2 inactivating mutation and alcohol consumption on Alzheimer's disease.

Aldehyde dehydrogenase 2 (ALDH2) is an enzyme found in the mitochondrial matrix that plays a central role in alcohol and aldehyde metabolism. A common ALDH2 polymorphism in East Asians descent (called ALDH2*2 or E504K missense variant, SNP ID: rs671), present in approximately 8% of the world's population, has been associated with a variety of diseases. Recent meta-analyses support the relationship between this ALDH2 polymorphism and Alzheimer's disease (AD). And AD-like pathology observed in ALDH2-/- null mice and ALDH2*2 overexpressing transgenic mice indicate that ALDH2 deficiency plays an important role in the pathogenesis of AD. Recently, the worldwide increase in alcohol consumption has drawn attention to the relationship between heavy alcohol consumption and AD. Of potential clinical significance, chronic administration of alcohol in ALDH2*2/*2 knock-in mice exacerbates the pathogenesis of AD-like symptoms. Therefore, ALDH2 polymorphism and alcohol consumption likely play an important role in the onset and progression of AD. Here, we review the data on the relationship between ALDH2 polymorphism, alcohol, and AD, and summarize what is currently known about the role of the common ALDH2 inactivating mutation, ALDH2*2, and alcohol in the onset and progression of AD.

Activation of aldehyde dehydrogenase-2 improves ischemic random skin flap survival in rats.

Objective: Random skin flaps have many applications in plastic and reconstructive surgeries. However, distal flap necrosis restricts wider clinical utility. Mitophagy, a vital form of autophagy for damaged mitochondria, is excessively activated in flap ischemia/reperfusion (I/R) injury, thus inducing cell death. Aldehyde dehydrogenase-2 (ALDH2), an allosteric tetrameric enzyme, plays an important role in regulating mitophagy. We explored whether ALDH2 activated by N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1) could reduce the risk of ischemic random skin flap necrosis, and the possible mechanism of action. Methods: Modified McFarlane flap models were established in 36 male Sprague-Dawley rats assigned randomly to three groups: a low-dose Alda-1 group (10 mg/kg/day), a high-dose Alda-1 group (20 mg/kg/day) and a control group. The percentage surviving skin flap area, neutrophil density and microvessel density (MVD) were evaluated on day 7. Oxidative stress was quantitated by measuring the superoxide dismutase (SOD) and malondialdehyde (MDA) levels. Blood perfusion and skin flap angiogenesis were assessed via laser Doppler flow imaging and lead oxide-gelatin angiography, respectively. The expression levels of inflammatory cytokines (IL-1ß, IL-6, and TNF-α), vascular endothelial growth factor (VEGF), ALDH2, PTEN-induced kinase 1 (PINK1), and E3 ubiquitin ligase (Parkin) were immunohistochemically detected. Indicators of mitophagy such as Beclin-1, p62, and microtubule-associated protein light chain 3 (LC3) were evaluated by immunofluorescence. Results: Alda-1 significantly enhanced the survival area of random skin flaps. The SOD activity increased and the MDA level decreased, suggesting that Alda-1 reduced oxidative stress. ALDH2 was upregulated, and mitophagy-related proteins (PINK1, Parkin, Beclin-1, p62, and LC3) were downregulated, indicating that ALDH2 inhibited mitophagy through the PINK1/Parkin signaling pathway. Treatment with Alda-1 reduced neutrophil infiltration and expressions of inflammatory cytokines. Alda-1 significantly upregulated VEGF expression, increased the MVD, promoted angiogenesis, and enhanced blood perfusion. Conclusion: ALDH2 activation can effectively enhance random skin flap viability via inhibiting PINK1/Parkin-dependent mitophagy. Moreover, enhancement of ALDH2 activity also exerts anti-inflammatory and angiogenic properties.

The administration of Alda-1, an activator of ALDH2, inhibits relapse-like ethanol intake in female alcohol-preferring UChB rats.

AIMS: Alcohol relapse is a main limitation for the treatment of alcohol use disorders. Previous studies have shown that Alda-1, a pharmacological activator of ALDH2, inhibits both acquisition and chronic ethanol intake in rats; however, its effects on relapse-like ethanol intake are unknown. The aim of this study was to assess the effect of Alda-1 on post-deprivation and reaccess relapse-like ethanol intake in alcohol-preferring UChB rats. We also aimed to assess the possible mechanisms associated with the effects of Alda-1 by measuring the levels of glutamate transporter (GLT-1), oxidative stress and neuroinflammation markers in different regions of the mesocorticolimbic system. MAIN METHODS: In Experiment I, UChB female rats were exposed for 100 days to voluntary ethanol intake followed by 2-weeks of ethanol withdrawal and 1 week of ethanol reaccess. Alda-1 (25 mg/kg, intragastric, i.g) or vehicle was administered daily for 14 days during the withdrawal/re-access period. Experiment II was similar to Experiment I, but after the withdrawal period, ethanol re-access was not allowed, and Alda-1 was administered during the last week of withdrawal. At the end of both experiments, the levels of GLT-1, oxidative stress (GSH, MDA), and neuroinflammation markers (GFAP, Iba-1) were assessed in nucleus accumbens and/or hippocampus. KEY FINDINGS: The results showed that Alda-1 administration markedly blocked (90 %, p < 0.001) relapse-like ethanol intake in UChB rats. Alda-1 increased Iba-1 reactivity (microglial marker) in the NAc of ethanol-deprived rats. Alda-1 administration did not influence the levels of GLT-1, oxidative stress markers (MDA, GSH) or GFAP reactivity in the mesocorticolimbic system. SIGNIFICANCE: These preclinical findings support the use of activators of ALDH2, such as Alda-1, as a potential pharmacological strategy in the treatment of alcohol relapse.

ALDH2 inhibition by lead and ethanol elicits redox imbalance and mitochondrial dysfunction in SH-SY5Y human neuroblastoma cell line: Reversion by Alda-1.

Lead (Pb), a common environmental contaminant, and ethanol (EtOH), a widely available drug of abuse, are well-known neurotoxicants. In vivo, experimental evidence indicates that Pb exposure affects oxidative EtOH metabolism with a high impact on living organisms. On these bases, we evaluated the consequences of combined Pb and EtOH exposure on aldehyde dehydrogenase 2 (ALDH2) functionality. In vitro exposure to 10 µM Pb, 200 mM EtOH, or their combination for 24 h reduced ALDH2 activity and content in SH-SY5Y human neuroblastoma cells. In this scenario, we observed mitochondrial dysfunction characterized by reduced mass and membrane potential, decreased maximal respiration, and spare capacity. We also evaluated the oxidative balance in these cells finding a significant increase in reactive oxygen species (ROS) production and lipid peroxidation products under all treatments accompanied by an increase in catalase (CAT) activity and content. These data suggest that ALDH2 inhibition induces the activation of converging cytotoxic mechanisms resulting in an interplay between mitochondrial dysfunction and oxidative stress. Notably, NAD+ (1 mM for 24 h) restored ALDH2 activity in all groups, while an ALDH2 enhancer (Alda-1, 20 µM for 24 h) also reversed some of the deleterious effects resulting from impaired ALDH2 function. Overall, these results reveal the crucial role of this enzyme on the Pb and EtOH interaction and the potential of activators such as Alda-1 as therapeutic approaches against several conditions involving aldehydes accumulation.

Ancestry component as a major predictor of lithium response in the treatment of bipolar disorder.

BACKGROUND: Bipolar Disorder (BD) represents the seventh major cause of disability life-years-adjusted. Lithium remains as a first-line treatment, but clinical improvement occurs only in 30 % of treated patients. Studies suggest that genetics plays a major role in shaping the individual response of BD patients to lithium. METHODS: We used machine-learning techniques (Advance Recursive Partitioned Analysis, ARPA) to build a personalized prediction framework of BD lithium response using biological, clinical, and demographical data. Using the Alda scale, we classified 172 BD I-II patients as responders or non-responders to lithium treatment. ARPA methods were used to build individual prediction frameworks and to define variable importance. Two predictive models were evaluated: 1) demographic and clinical data, and 2) demographic, clinical and ancestry data. Model performance was assessed using Receiver Operating Characteristic (ROC) curves. RESULTS: The predictive model including ancestry yield the best performance (sensibility = 84.6 %, specificity = 93.8 % and AUC = 89.2 %) compared to the model without ancestry (sensibility = 50 %, Specificity = 94.5 %, and AUC = 72.2 %). This ancestry component best predicted lithium individual response. Clinical variables such as disease duration, the number of depressive episodes, the total number of affective episodes, and the number of manic episodes were also important predictors. CONCLUSION: Ancestry component is a major predictor and significantly improves the definition of individual Lithium response in BD patients. We provide classification trees with potential bench application in the clinical setting. While this prediction framework might be applied in specific populations, the used methodology might be of general use in precision and translational medicine.

A mixed-methods program evaluation of the Alda Healthcare Experience- a program to improve healthcare team communication.

BACKGROUND: Communication among interprofessional healthcare worker teams is critical to ensure a thriving and resilient workforce. We will evaluate the implementation and effectiveness of the Alda Healthcare Experience (AHE), a novel medical improvisation (improv) workshop designed to improve interprofessional communication skills among healthcare professionals. The AHE workshop includes a two-hour experiential training workshop led by an improv specialist and a clinical co-facilitator. In July 2022 we began implementing the AHE workshop by training 18 clinical co-facilitators who will co-facilitate the workshops for 550 healthcare workers from five hospital departments at Stony Brook University Hospital over the course of a year and a half. Using mixed-methods, we will conduct an Effectiveness-Implementation Hybrid Design project that includes an outcome evaluation (effectiveness) and a process evaluation (implementation). METHODS: Our outcome evaluation will assess the impact of the AHE workshop on short- and long-term improvement in interprofessional communication, stress, and professional fulfillment. The process evaluation component will examine programmatic, organizational, and individual facilitators or barriers to effective implementation of the AHE workshop. Qualitative methods will include dimensional analysis employing individual interviews of 20-40 AHE Project Participants, 5-10 Selected Informants, and all the clinical co-facilitators. Quantitative methods will use a quasi-experimental longitudinal design with an intervention group and surveillance of a control group (wait-list) and repeated assessments using validated instruments measuring communications skills, professional fulfillment, stress, burnout, uncertainty tolerance, and teamwork. DISCUSSION: Effective and efficient communication within healthcare teams is fundamental to building team cohesion that, in turn, supports individual resilience and builds positive organizational culture. The AHE program is an innovative approach to improve interprofessional healthcare communication and reduce healthcare worker burnout. In addition to institutional buy-in, rigorous evaluations of medical improv programs are necessary as a critical step in making such programs scalable. TRIAL REGISTRATION: N/A.

The Loss-of-Function Mutation aldA67 Leads to Enhanced α-L-Rhamnosidase Production by Aspergillus nidulans.

In Aspergillus nidulans L-rhamnose is catabolised to pyruvate and L-lactaldehyde, and the latter ultimately to L-lactate, via the non-phosphorylated pathway (LRA) encoded by the genes lraA-D, and aldA that encodes a broad substrate range aldehyde dehydrogenase (ALDH) that also functions in ethanol utilisation. LRA pathway expression requires both the pathway-specific transcriptional activator RhaR (rhaR is expressed constitutively) and the presence of L-rhamnose. The deletion of lraA severely impairs growth when L-rhamnose is the sole source of carbon and in addition it abolishes the induction of genes that respond to L-rhamnose/RhaR, indicating that an intermediate of the LRA pathway is the physiological inducer likely required to activate RhaR. The loss-of-function mutation aldA67 also has a severe negative impact on growth on L-rhamnose but, in contrast to the deletion of lraA, the expression levels of L-rhamnose/RhaR-responsive genes under inducing conditions are substantially up-regulated and the production of α-L-rhamnosidase activity is greatly increased compared to the aldA+ control. These findings are consistent with accumulation of the physiological inducer as a consequence of the loss of ALDH activity. Our observations suggest that aldA loss-of-function mutants could be biotechnologically relevant candidates for the over-production of α-L-rhamnosidase activity or the expression of heterologous genes driven by RhaR-responsive promoters.

Aldehyde Dehydrogenase 2 Activator Augments the Beneficial Effects of Empagliflozin in Mice with Diabetes-Associated HFpEF.

To ameliorate diabetes mellitus-associated heart failure with preserved ejection fraction (HFpEF), we plan to lower diabetes-mediated oxidative stress-induced 4-hydroxy-2-nonenal (4HNE) accumulation by pharmacological agents that either decrease 4HNE generation or increase its detoxification.A cellular reactive carbonyl species (RCS), 4HNE, was significantly increased in diabetic hearts due to a diabetes-induced decrease in 4HNE detoxification by aldehyde dehydrogenase (ALDH) 2, a cardiac mitochondrial enzyme that metabolizes 4HNE. Therefore, hyperglycemia-induced 4HNE is critical for diabetes-mediated cardiotoxicity and we hypothesize that lowering 4HNE ameliorates diabetes-associated HFpEF. We fed a high-fat diet to ALDH2*2 mice, which have intrinsically low ALDH2 activity, to induce type-2 diabetes. After 4 months of diabetes, the mice exhibited features of HFpEF along with increased 4HNE adducts, and we treated them with vehicle, empagliflozin (EMP) (3 mg/kg/d) to reduce 4HNE and Alda-1 (10 mg/kg/d), and ALDH2 activator to enhance ALDH2 activity as well as a combination of EMP + Alda-1 (E + A), via subcutaneous osmotic pumps. After 2 months of treatments, cardiac function was assessed by conscious echocardiography before and after exercise stress. EMP + Alda-1 improved exercise tolerance, diastolic and systolic function, 4HNE detoxification and cardiac liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathways in ALDH2*2 mice with diabetes-associated HFpEF. This combination was even more effective than EMP alone. Our data indicate that ALDH2 activation along with the treatment of hypoglycemic agents may be a salient strategy to alleviate diabetes-associated HFpEF.

Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: The lipid oxidation is a key factor for damaging hepatocytes and causing cell death. However, the mechanisms underlying hepatocyte death and the role of the most popular lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in nonalcoholic steatohepatitis (NASH) remains unclear. METHODS: We demonstrated using hepatoma cell lines, a NASH mouse model, HNE-treated monkeys, and biopsy specimens from patients with NASH that HNE induced hepatocyte death by disintegrating the lysosomal limiting membrane. RESULTS: The degree of HNE deposition in human NASH hepatocytes was more severe in cases with high lobular inflammation, ballooning, and fibrosis scores, and was associated with enlargement of the staining of lysosomes in hepatocytes. In in vitro experiments, HNE activated µ-calpain via G-protein coupled receptor (GPR) 120. The resultant rupture/permeabilization of the lysosomal limiting membrane induced the leakage of cathepsins from lysosomes and hepatocyte death. The blockade of G-protein coupled receptor 120 (GPR120) or µ-calpain expression suppressed lysosomal membrane damage and hepatocyte death by HNE. Alda-1, which activates aldehyde dehydrogenase 2 to degrade HNE, prevented HNE-induced hepatocyte death. Intravenous administration of HNE to monkeys for 6 months resulted in hepatocyte death by a mechanism similar to that of cultured cells. In addition, intraperitoneal administration of Alda-1 to choline-deficient, amino-acid defined treated mice for 8 weeks inhibited HNE deposition, decreased liver inflammation, and disrupted lysosomal membranes in hepatocytes, resulting in improvement of liver fibrosis. CONCLUSIONS: These results provide novel insights into the mechanism of hepatocyte death in NASH and will contribute to the development of new therapeutic strategies for NASH.

The Effects of Alda-1 Treatment on Renal and Intestinal Injuries After Cardiopulmonary Resuscitation in Pigs.

Aim: After successful cardiopulmonary resuscitation (CPR), most survivors will develop acute kidney injury and intestinal barrier dysfunction, both of which contribute to the poor outcomes of cardiac arrest (CA) victims. Recently, the aldehyde dehydrogenase 2 (ALDH2) agonist, Alda-1 was shown to effectively alleviate regional ischemia/reperfusion injury of various organs. In the present study, we investigated the effects of Alda-1 treatment on renal and intestinal injuries after CA and resuscitation in pigs. Methods: Twenty-four male domestic pigs were randomly divided into one of the three groups: sham (n = 6), CPR (n = 10), or CPR+Alda-1 (n = 8). CA was induced and untreated for 8 min, and then CPR was performed for 8 min in the CPR and CPR+Alda-1 groups. At 5 min after resuscitation, a dose of 0.88 mg/kg of Alda-1 was intravenously administered in the CPR+Alda-1 group. The biomarkers of renal and intestinal injuries after resuscitation were regularly measured for a total of 24 h. Subsequently, the animals were euthanized, and then renal and intestinal tissues were obtained for the measurements of ALDH2 activity and expression, and cell apoptosis and ferroptosis. Results: Five of the 10 animals in the CPR group and six of the eight animals in the CPR+Alda-1 group were successfully resuscitated. After resuscitation, the levels of biomarkers of renal and intestinal injuries were significantly increased in all animals experiencing CA and resuscitation compared with the sham group; however, Alda-1 treatment significantly alleviated renal and intestinal injuries compared to the CPR group. Post-resuscitation ALDH2 activity was significantly decreased and its expression was markedly reduced in the kidney and intestine in those resuscitated animals compared with the sham group; nevertheless, both of them were significantly greater in those animals receiving Alda-1 treatment compared to the CPR group. In addition, renal, intestinal apoptosis and ferroptosis after resuscitation were observed in the CPR and CPR+Alda-1 groups, in which both of them were significantly milder in the CPR+Alda1 group than in the CPR group. Conclusions: The activation of ALDH2 by Alda-1 treatment significantly alleviated post-resuscitation renal and intestinal injuries through the inhibition of cell apoptosis and ferroptosis in a pig model of CA and resuscitation.

Is Poor Lithium Response in Individuals with Bipolar Disorder Associated with Increased Degradation of Tryptophan along the Kynurenine Pathway? Results of an Exploratory Study.

Bipolar disorder is associated with an inflammation-triggered elevated catabolism of tryptophan to the kynurenine pathway, which impacts psychiatric symptoms and outcomes. The data indicate that lithium exerts anti-inflammatory effects by inhibiting indoleamine-2,3-dioxygenase (IDO)-1 activity. This exploratory study aimed to investigate the tryptophan catabolism in individuals with bipolar disorder (n = 48) compared to healthy controls (n = 48), and the associations with the response to mood stabilizers such as lithium, valproate, or lamotrigine rated with the Retrospective Assessment of the Lithium Response Phenotype Scale (or the Alda scale). The results demonstrate an association of a poorer response to lithium with higher levels of kynurenine, kynurenine/tryptophan ratio as a proxy for IDO-1 activity, as well as quinolinic acid, which, overall, indicates a pro-inflammatory state with a higher degradation of tryptophan towards the neurotoxic branch. The treatment response to valproate and lamotrigine was not associated with the levels of the tryptophan metabolites. These findings support the anti-inflammatory properties of lithium. Furthermore, since quinolinic acid has neurotoxic features via the glutamatergic pathway, they also strengthen the assumption that the clinical drug response might be associated with biochemical processes. The relationship between the lithium response and the measurements of the tryptophan to the kynurenine pathway is of clinical relevance and may potentially bring advantages towards a personalized medicine approach to bipolar disorder that allows for the selection of the most effective mood-stabilizing drug.

Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice.

Ethanol increases hepatic mitophagy driven by unknown mechanisms. Type 1 mitophagy sequesters polarized mitochondria for nutrient recovery and cytoplasmic remodeling. In Type 2, mitochondrial depolarization (mtDepo) initiates mitophagy to remove the damaged organelles. Previously, we showed that acute ethanol administration produces reversible hepatic mtDepo. Here, we tested the hypothesis that ethanol-induced mtDepo initiates Type 2 mitophagy. GFP-LC3 transgenic mice were gavaged with ethanol (2-6 g/kg) with and without pre-treatment with agents that decrease or increase mtDepo-Alda-1, tacrolimus, or disulfiram. Without ethanol, virtually all hepatocytes contained polarized mitochondria with infrequent autophagic GFP-LC3 puncta visualized by intravital microscopy. At ~4 h after ethanol treatment, mtDepo occurred in an all-or-none fashion within individual hepatocytes, which increased dose dependently. GFP-LC3 puncta increased in parallel, predominantly in hepatocytes with mtDepo. Mitochondrial PINK1 and PRKN/parkin also increased. After covalent labeling of mitochondria with MitoTracker Red (MTR), GFP-LC3 puncta encircled MTR-labeled mitochondria after ethanol treatment, directly demonstrating mitophagy. GFP-LC3 puncta did not associate with fat droplets visualized with BODIPY558/568, indicating that increased autophagy was not due to lipophagy. Before ethanol administration, rhodamine-dextran (RhDex)-labeled lysosomes showed little association with GFP-LC3. After ethanol treatment, TFEB (transcription factor EB) translocated to nuclei, and lysosomal mass increased. Many GFP-LC3 puncta merged with RhDex-labeled lysosomes, showing autophagosomal processing into lysosomes. After ethanol treatment, disulfiram increased, whereas Alda-1 and tacrolimus decreased mtDepo, and mitophagy changed proportionately. In conclusion, mtDepo after acute ethanol treatment induces mitophagic sequestration and subsequent lysosomal processing.Abbreviations : AcAld, acetaldehyde; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ALD, alcoholic liver disease; Alda-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; LMNB1, lamin B1; MAA, malondialdehyde-acetaldehyde adducts; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MPT, mitochondrial permeability transition; mtDAMPS, mitochondrial damage-associated molecular patterns; mtDepo, mitochondrial depolarization; mtDNA, mitochondrial DNA; MTR, MitoTracker Red; PI, propidium iodide; PINK1, PTEN induced putative kinase 1; PRKN, parkin; RhDex, rhodamine dextran; TFEB, transcription factor EB; Tg, transgenic; TMRM, tetramethylrhodamine methylester; TOMM20, translocase of outer mitochondrial membrane 20; VDAC, voltage-dependent anion channel.

Methylomic Biomarkers of Lithium Response in Bipolar Disorder: A Proof of Transferability Study.

Response to lithium (Li) is highly variable in bipolar disorders (BD) and no clinical or biological predictors of long-term response have been validated to date. Using a genome-wide methylomic approach (SeqCapEpi), we previously identified seven differentially methylated regions (DMRs) that discriminated good from non-responders (prophylactic response phenotype defined using the "Alda" scale). This study is a proof of transferability from bench to bedside of this epigenetic signature. For this purpose, we used Methylation Specific High-Resolution Melting (MS-HRM), a PCR based method that can be implemented in any medical laboratory at low cost and with minimal equipment. In 23 individuals with BD, MS-HRM measures of three out of seven DMRs were technically feasible and consistencies between SeqCapEpi and MS-HRM-measures were moderate to high. In an extended sample of individuals with BD (n = 70), the three MS-HRM-measured DMRs mainly predicted nonresponse, with AUC between 0.70-0.80 according to different definitions of the phenotype (Alda- or machine-learning-based definitions). Classification tree analyses further suggested that the MS-HRM-measured DMRs correctly classified up to 84% of individuals as good or non-responders. This study suggested that epigenetic biomarkers, identified in a retrospective sample, accurately discriminate non-responders from responders to Li and may be transferrable to routine practice.

Alda-1, an Activator of ALDH2, Improves Postresuscitation Cardiac and Neurological Outcomes by Inhibiting Pyroptosis in Swine.

Aldehyde dehydrogenase 2 (ALDH2) has been proven to protect the heart and brain against regional ischemia/reperfusion injury, in which the protective role is related to the inhibition of pyroptosis. In the present study, we investigated whether an ALDH2 activator N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichloro-benzamide (Alda-1) would improve postresuscitation cardiac and neurological outcomes in a clinically relevant swine model of cardiac arrest (CA) and resuscitation. The animal model was established by 8 min of untreated ventricular fibrillation and then 8 min of cardiopulmonary resuscitation (CPR). After restoring spontaneous circulation, the animals were randomly divided to receive either Alda-1 (0.88 mg/kg, n = 6) or saline (n = 5). Postresuscitation hemodynamic parameters, cardiac function, and cardiac and cerebral injuries were periodically measured for a total of 24 h. At 24 h postresuscitation, neurological function was evaluated, and then the animals were sacrificed, and cardiac and cerebral tissue samples were obtained for the measurements of oxidative stress, inflammation and pyroptosis. Consequently, postresuscitation cardiac and neurological dysfunction were significantly improved accompanied with significantly milder cardiac and cerebral injuries in the Alda-1 group compared with the CPR group. In addition, the increase in NLR family pyrin domain-containing 3 inflammasome expression and proinflammatory cytokine production, which indicated the occurrence of inflammatory response, were significantly less in the Alda1 group than in the CPR group. The expression level of gasdermin D used as a protein marker of pyroptosis was also significantly reduced in all resuscitated animals receiving Alda1 treatment. Moreover, the severity of oxidative stress indicated by the changes of 4-hydroxy-2-nonenal and malondialdehyde was significantly decreased in the heart and brain in all animals treated with Alda-1 compared to the CPR group. Thus, Alda-1 mitigated postresuscitation cardiac and neurological dysfunction and injuries possibly by inhibiting oxidative stress-mediated NLRP3 inflammasome activation and pyroptosis in a swine model of CA and resuscitation.

In Silico Docking of Novel Phytoalkaloid Camalexin in the Management of Benomyl Induced Parkinson's Disease and its In Vivo Evaluation by Zebrafish Model.

BACKGROUND: Parkinson's Disease (PD) exhibits the extrapyramidal symptoms caused due to the dopaminergic neuronal degeneration in the substantia nigra of the brain and depletion of Aldehyde Dehydrogenase (ALDH) enzyme. OBJECTIVE: This study was designed to enlighten the importance of the Aldehyde dehydrogenase enzyme in protecting the dopamine levels in a living system. Camalexin, a potentially active compound, has been evaluated for its dopamine enhancing and aldehyde dehydrogenase protecting role in pesticide-induced Parkinson's disease. METHODS: AutoDock 4.2 software was employed to perform the docking simulations between the ligand camalexin and standard drugs Alda-1, Ropirinole with three proteins 4WJR, 3INL, 5AER. Consequently, the compound was evaluated for its in vivo neuroprotective role in the zebrafish model by attaining Institutional Animal Ethical Committee permission. The behavioral assessments and catecholamine analysis in zebrafish were performed. RESULTS: The Autodock result shows that the ligand camalexin has a lower binding energy (-3.84) that indicates a higher affinity with the proteins when compared to the standard drug of proteins (-3.42). In the zebrafish model, behavioral studies provided evidence that camalexin helps in the improvement of motor functions and cognition. The catecholamine assay has proved that there is an enhancement in dopamine levels, as well as an improvement in aldehyde dehydrogenase enzyme. CONCLUSION: The novel compound, camalexin, offers a protective role in Parkinson's disease model by its interaction with neurochemical proteins and also in alternative in vivo model.

Effect of Alda-1 on ferroptosis in cardiomyocytes after cardiac arrest and cardiopulmonary resuscitation in swine / 中华麻醉学杂志

Objective:To evaluate the effect of Alda-1 on ferroptosis in cardiomyocytes after cardiac arrest and cardiopulmonary resuscitation in swine.Methods:Twenty-two healthy male white swine, weighing 35-43 kg, were divided into 3 groups using a random number table method: sham operation group (group S, n=6), cardiac arrest-cardiopulmonary resuscitation group (group CA-CPR, n=8) and Alda-1 group ( n=8). The animals only underwent the general preparation in group S, and the swine model of cardiac arrest and cardiopulmonary resuscitation was developed by 8 min of electrically induced cardiac arrest through the pacing catheter in the right ventricle followed by 8 min of cardiopulmonary resuscitation in CA-CPR and Alda-1 groups.Alda-1 0.88 mg/kg was intravenously injected at 5 min after resuscitation in group Alda-1, and the equal volume of vehicle was administered instead in the other two groups.Stroke volume (SV) and global ejection fraction (GEF) were measured using PiCCO before developing the model and at 1, 2 and 4 h after resuscitation (T 0-3). Venous blood samples were collected from the femoral vein to measure the concentrations of serum cardiac troponin (cTnI) by enzyme-linked immunosorbent assay at T 0-3, and at 24 h after resuscitation (T 4). The animals were then sacrificed, and myocardial tissues in the left ventricle were harvested to measure the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4) (by Western blot), iron deposition (by Prussian blue staining), 4-hydroxy-2-nonenal (4-HNE) content (by enzyme-linked immunosorbent assay), and malondialdehyde (MDA) and glutathione (GSH) contents (by colorimetry). Results:Compared with group S, SV and GEF were significantly decreased at T 1-3, the serum concentrations of cTnI were increased at T 1-4, myocardial ACSL4 expression was up-regulated, GPX4 expression was down-regulated, iron deposition and contents of 4-HNE and MDA were increased, and the content of GSH was decreased in CA-CPR and Alda-1 groups ( P<0.05). Compared with group CA-CPR, SV and GEF were significantly increased at T 2-3, the serum concentrations of cTnI were decreased at T 3-4, myocardial ACSL4 expression was down-regulated, GPX4 expression was up-regulated, iron deposition and contents of 4-HNE and MDA were decreased, and the content of GSH was increased in group Alda-1 ( P<0.05). Conclusions:Alda-1 can alleviate myocardial injury after cardiac arrest and cardiopulmonary resuscitation in swine and further improve cardiac dysfunction, and the mechanism may be related to inhibition of cell ferroptosis.

Critical role of mitochondrial aldehyde dehydrogenase 2 in acrolein sequestering in rat spinal cord injury.

Lipid peroxidation-derived aldehydes, such as acrolein, the most reactive aldehyde, have emerged as key culprits in sustaining post-spinal cord injury (SCI) secondary pathologies leading to functional loss. Strong evidence suggests that mitochondrial aldehyde dehydrogenase-2 (ALDH2), a key oxidoreductase and powerful endogenous anti-aldehyde machinery, is likely important for protecting neurons from aldehydes-mediated degeneration. Using a rat model of spinal cord contusion injury and recently discovered ALDH2 activator (Alda-1), we planned to validate the aldehyde-clearing and neuroprotective role of ALDH2. Over an acute 2 day period post injury, we found that ALDH2 expression was significantly lowered post-SCI, but not so in rats given Alda-1. This lower enzymatic expression may be linked to heightened acrolein-ALDH2 adduction, which was revealed in co-immunoprecipitation experiments. We have also found that administration of Alda-1 to SCI rats significantly lowered acrolein in the spinal cord, and reduced cyst pathology. In addition, Alda-1 treatment also resulted in significant improvement of motor function and attenuated post-SCI mechanical hypersensitivity up to 28 days post-SCI. Finally, ALDH2 was found to play a critical role in in vitro protection of PC12 cells from acrolein exposure. It is expected that the outcome of this study will broaden and enhance anti-aldehyde strategies in combating post-SCI neurodegeneration and potentially bring treatment to millions of SCI victims. All animal work was approved by Purdue Animal Care and Use Committee (approval No. 1111000095) on January 1, 2021.